EAGER: Repair and Recombination of Mitochondrial DNA

DNA损伤，特别是以双链染色体断裂（DSBs）的形式，对基因组的完整性构成严重威胁。关于真核细胞通过同源定向修复和非同源末端连接过程应对DSB损伤的各种方式，我们已经了解了很多，但有一个值得注意的例外：实际上，我们对线粒体DNA是如何修复的一无所知，无论是在人类身上，还是在研究得最好的模式生物——酿酒酵母身上。了解DSB修复和重组如何在线粒体中发生，可能为许多与mtDNA缺陷相关的人类线粒体疾病的基因治疗方法打开大门。该项目还为博士后学者和本科生提供研究和职业发展机会。出芽酵母线粒体DNA （mtDNA）在单倍体结合时发生频繁的同源重组，亲本双方都将mtDNA贡献给受精卵；但几乎不知道这些事件是如何开始的，或者通过什么中间体进行重组。此外，酵母线粒体表现出非常高水平的由位点特异性核酸内切酶I-SceI启动的单向遗传转移（基因转换）；但这些事件是如何发生的尚不清楚，除了它们在很大程度上独立于核内染色体之间发生的类似基因转换过程所必需的基因，包括由同一内切酶启动的DSB修复事件。这个EAGER项目的目标有三个：1)通过CRISPRi（通过条件的、显性的dcas9介导的抑制基因表达）对出芽酵母进行全基因组筛选，以鉴定mtdna之间一般同源重组所需的基因；2)鉴定位点特异性I-SceI内切酶介导的基因转化所需的特异性基因；3)在酵母中寻找允许DNA摄取的突变体，以影响线粒体内的基因编辑。最后一个目标解决了一个艰巨的挑战：到目前为止，还不可能将可用于基因编辑的DNA引入线粒体（例如通过cas9介导的模板修复）。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

DNA damage, especially in the form of double-strand chromosome breaks (DSBs), poses a severe threat to genome integrity. Much has been learned about the various ways that eukaryotic cells can cope with DSB damage, through both homology-directed repair and nonhomologous end-joining processes, with one notable exception: virtually nothing is known about how DNA in mitochondria is repaired, in humans or even in the best-studied model organism, Saccharomyces cerevisiae. Understanding of how DSB repair and recombination occur in mitochondria may open the door to gene therapeutic approaches for the many human mitochondrial diseases that are related to defects in mtDNA. The project also provides research and career development opportunities for a post-doctoral scholar and undergraduate student.Budding yeast mitochondrial DNA (mtDNA) undergoes frequent homologous recombination when haploids conjugate and both parents contribute mtDNA to the zygote; but almost nothing is known about how these events are initiated or through what intermediates recombination progresses. In addition, yeast mitochondria exhibit very high levels of unidirectional genetic transfer (gene conversion) initiated by the site-specific endonuclease, I-SceI; but how such events occur is unknown, save for the fact that they are largely independent of the genes essential for analogous gene conversion processes occurring between chromosomes in the nucleus, including DSB repair events initiated by the same endonuclease. The goals of this EAGER project are threefold: 1) to use genome-wide screening of budding yeast by CRISPRi (inhibition of gene expression by conditional, dominant dCas9-mediated repression) to identify genes necessary for general homologous recombination between mtDNAs; 2) to identify specifically genes that are required for site-specific I-SceI endonuclease- mediated gene conversion; and 3) to seek mutants in yeast that would allow the uptake of DNA to effect gene editing within mitochondria. The last aim tackles a formidable challenge: to date it has not been possible to introduce DNA into the mitochondrion that could be used for gene editing (e.g. by Cas9-mediated template repair).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.